Electrosonic cell manipulation device

ABSTRACT

In method of injecting a substance into a living cell having a cell membrane, the substance, the cell and a liquid are placed into a tapering passage. Energy is applied to the cell, thereby inducing poration. To sort cells, a cellular suspension is placed in a tapering passage, including a narrow end that defines an opening that has a dimension corresponding to a cell size. An acoustic wave is applied, thereby forcing cells having a cell size smaller than the selected cell size through the opening, with a portion of the cells having a cell size not smaller than the selected cell size not forced through the opening. To extract material from a cell, an electric field and an acoustic wave are applied, thereby causing the cell membrane to allow the material to pass out of the cell.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/666,661, filed Mar. 30, 2005, the entirety ofwhich is hereby incorporated herein by reference.

This application is a divisional of, and claims the benefit of, U.S.patent application Ser. No. 11/277,662, filed Mar. 28, 2006, now issuedas U.S. Pat. No. 7,704,743, the entirety of which is hereby incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to cellular manipulation devices and, morespecifically, to a device that can perform poration, transfection, lysisand sorting of living cells.

2. Description of the Related Art

As reflected in the recent Proteomics special feature article(“Automated NanoElectrospray: A New Advance for Proteomics Researchers,”Laboratory News, 2002) Mass Spectrometry (MS) has become the technologyof choice to meet today's unprecedented demand for accuratebioanalytical measurements, including protein identification. AlthoughMS can be used to analyze biomolecules with very large molecular weights(up to several MegaDaltons (Mda)), these molecules must be firstconverted to gas-phase ions before they can be introduced into a massspectrometer for analysis. Electrospray ionization (ESI) has proven tobe an enormous breakthrough in structural biology because it provides amechanism for transferring large biological molecules into the gas phaseas intact charged ions. It is the creation of efficient conversion of avery small quantity of a liquid sample (proteins are very expensive andoften very difficult to produce in sizable quantities) into gas-phaseions that is one of the main bottlenecks for using mass spectrometry inhigh throughput proteomics.

Conventional (micro and nano) capillary ESI sources, as well as the morerecently developed MEMS-based electrospray devices, rely on applicationof strong electric field, which is used for focusing of the charged jetleading to jet tip instabilities and formation of small droplets of theanalyte sample. As a result, the size and homogeneity of the formeddroplets is determined by the magnitude and geometry of the appliedelectric field, thus requiring high voltages for generating sufficientlysmall micrometer or sub-micrometer droplets via the so-called Taylorcone nebulization. Reliance on the electrohydrodynamic Taylor conefocusing of the jet to form the mist of sufficiently small chargeddroplets leading to single ion formation imposes several fundamental andsignificant limitations on the capabilities of the conventional ESIinterface.

One such problem is that a very large electric potential needs to beapplied to the capillary tip (up to a few kilovolts relative to theground electrode of the MS interface) to ensure formation of the stableTaylor cone, especially at higher flow rates and with poorly conductingorganic solvents.

An additional problem is that the choice of suitable solvents is verymuch restricted to those featuring high electrical conductivity andsufficiently low surface tension. This restriction imposes severelimitations on the range of biological molecules that can be analyzedvia ESI Mass Spectrometry. For example, use of pure water (the mostnatural environment for most biomolecules) as a solvent is difficult inconventional ESI since the required onset electrospray voltage isgreater than that of the corona discharge, leading to an unstable Taylorcone, damage to the emitter and uncontrollable droplet/ion formation.

Since the conventional ESI relies on the disintegration of thecontinuous jet emanating from the Taylor cone into an aerosol of chargeddroplets, there is the limit to the lowest flow rate (and therefore theminimum sample size) that can be used during the analysis. For example,commercial products require the minimum sample volume to be about 3 μL.

Another problem is that sample utilization (i.e., fraction of the samplevolume that is introduced and being used in MS analysis relative to thetotal volume of the electrosprayed sample) is very low due touncontrollable nature of electrohydrodynamic atomization process thatrelies on the surface instabilities. Further, a significant dead volume(i.e., a fraction of the sample that cannot be pulled from the capillaryby electrical forces) is unavoidable in any jet-based atomizationprocess.

Still other problems are that commercially available ESI devices arevery expensive because of the manufacturing difficulties, and limitedusable lifetime because of the high voltage operation in achemically-aggressive solvent environment.

An ability to extract DNA from or inject DNA into living cells iscritical to any genetic, molecular biology, drug design and delivery,and pharmaceutical research and development work. Drug delivery,pharmaceutical, and biotech industries routinely need to be able toextract DNA from and inject DNA into a cell. This is probably the mostcritical step in many molecular biology and genetics modificationprotocols currently used.

Some methods of injecting DNA into cells involve poration of a group ofcells. In poration, the cells are subjected to an energy field thatcauses pores in the cell membranes to dilate. Typically, many cells areplaced in a field that varies spatially and those cells that are in thearea of a certain field strength porate, while the rest do not. The lowlevel of predictability and accuracy of poration results in a low yieldand the inefficiency of requiring the technician to spend extra timesorting cells that have successfully porated from those that have notsuccessfully porated.

Therefore, there is a need for a system for extracting and injectingmaterials into living cells with a high level of predictability andaccuracy.

SUMMARY OF THE INVENTION

The disadvantages of the prior art are overcome by the present inventionwhich, in one aspect, is a method of injecting a substance into a livingcell having a cell membrane. The substance, the cell and a liquid areplaced into a tapering passage. An energy is applied to the cellsufficient to induce poration of the cell.

In another aspect, the invention is a method of sorting cells, in whichthe cells are suspended in a liquid, thereby creating a cellularsuspension. The cellular suspension is placed in a tapering passage. Thetapering passage includes a wide end and an oppositely-disposed narrowend that defines an opening, with the opening having a dimensioncorresponding to a selected cell size. A standing acoustic wave isapplied to the cells, thereby forcing cells having a cell size smallerthan the selected cell size through the opening and so that at least aportion of the cells having a cell size not smaller than the selectedcell size are not forced through the opening.

In another aspect, the invention is a method of extracting material froma cell, having a cell membrane, in which the cell is suspended in aliquid, thereby creating a cellular suspension. A predetermined electricfield is applied to the cell. An acoustic wave is applied to the cell.The electric field and the acoustic wave cause the cell membrane toallow the material to pass out of the cell.

In yet another aspect, the invention is an apparatus for manipulatingcells that includes a substrate, a first poration electrode, a secondporation electrode, a fluid driving structure and an oscillatingcircuit. The substrate has a first side and an opposite second side anddefines at least one tapering passage passing therethrough. The taperingpassage opens to the first side with a wide end and also opens to thesecond side with a narrow end. The narrow end has a size thatcorresponds to a predetermined characteristic of a selected cell. Thefirst poration electrode is spaced-apart from the second porationelectrode and is disposed so as to impart a predetermined electricalfield on the passage when an electrical potential is applied between thefirst poration electrode and the second poration electrode. The fluiddriving structure drives fluid through the opening. The oscillatingcircuit applies an oscillating potential to the ultrasonic transducer,thereby causing the ultrasonic transducer to generate a standing wave inthe tapering passage. The standing wave and the electrical field impartenergy on at least a portion of the cells so as to cause a predeterminedaction on the cells.

A device for on-demand DNA delivery in or out of the cell via acombination (or possibly individual action) of ultrasonic and electricalporation or lysis, respectively, of the cell membrane is disclosed. Inaddition to poration and lysing functionality, the device also includesthe capability of in-line size selective cell sorting (via control ofthe ejector nozzle size) prior to poration or lysis. It also enablestransport of modified cell DNA to a final destination as apost-poration/lysis step for further processing. The device can operatein both high-throughput and multiplexed mode in the microarray format.

These and other aspects of the invention will become apparent from thefollowing description of the preferred embodiments taken in conjunctionwith the following drawings. As would be obvious to one skilled in theart, many variations and modifications of the invention may be effectedwithout departing from the spirit and scope of the novel concepts of thedisclosure.

BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWINGS

FIG. 1 is a is a schematic of a representative embodiment of a massspectrometry system.

FIG. 2 is an illustration of a cross-section of an embodiment of anelectrospray system, as shown in FIG. 1.

FIG. 3 is an illustration of a cross-section of another embodiment of anelectrospray system, as shown in FIG. 1.

FIGS. 4A-4J are illustrations of cross-sections of a representativeembodiment of a method of forming the electrospray system shown in FIG.3.

FIG. 5 is an illustration of a cross-section of another embodiment of anelectrospray system, as shown in FIG. 1.

FIG. 6 is an illustration of a cross-section of another embodiment of anelectrospray system, as shown in FIG. 1.

FIG. 7 is an illustration of a cross-section of another embodiment of anelectrospray system, as shown in FIG. 1.

FIGS. 8A-8K are illustrations of cross-sections of a representativeembodiment of a method of forming the electrospray system shown in FIG.7.

FIGS. 9A-9D are illustrations of top views of representative embodimentsof an electrospray system. FIG. 9B illustrates an acousticallyresponsive fluid bubble in one section of the electrospray system, whileFIG. 9C illustrates a fluid bubble in the other section of theelectrospray system.

FIGS. 10A-10F are illustrations of top views of representativeembodiments of an electrospray system. FIGS. 10B through 10F illustratean acoustically responsive fluid bubble being positioned from onesection of the electrospray system to another.

FIG. 11 is a schematic of a representative micro-machined ultrasonicdroplet generator.

FIG. 12 is a schematic of a representative process for forming themicro-machined ultrasonic droplet generator illustrated in FIG. 11.

FIGS. 13A-13B illustrate scanning electron micrographs (SEMs) of aKOH-etched pyramid-shaped horn with an ICP etched nozzle at the apex(FIG. 13A) and an array of nozzles fabricated on a silicon wafer (FIG.13B).

FIG. 14A illustrates a droplet ejection from several nozzles of aprototype device.

FIG. 14B illustrates a stroboscopic image of a jet of about 8 μmdiameter droplets ejected by a representative electrospray system.

FIG. 14C illustrates a stroboscopic image of a jet of 5 μm dropletsejected by a representative electrospray system.

FIG. 15 illustrates a schematic of a representative experimental setupfor experimental characterization of the micro-machined ultrasonicelectrospray array when interfaced with a mass spectrometer (MS).

FIG. 16 illustrates an MS spectra of the MeOH:H20:Acetic Acid(50:49.9:0.1) solvent mixture containing a standard low molecular weighttest compound reserpine (MW=609 Da, CAS#50-55-5) ionized using theelectrospray system.

FIG. 17A is a cross-sectional view of one embodiment that may beemployed cell manipulation.

FIG. 17B is a cross-sectional view of a second embodiment that may beemployed cell manipulation.

FIG. 18A is a plan view of an embodiment employing an array of taperingpassages.

FIG. 18B is a plan view showing an opposite side of the embodiment shownin FIG. 18A.

FIG. 18C is a cross-sectional view of the embodiment shown in FIG. 18B,taken along line 18C-18C.

FIG. 19 is a micrograph of a tapering passage.

FIG. 20A is a schematic diagram showing the filling of tapering passageswith differing materials.

FIG. 20B is a schematic diagram showing the poration of the materialsshown in FIG. 20A.

FIG. 21 is a cross-sectional view of an embodiment employing a fluidpump.

FIG. 22 is a plan view of an embodiment employing a planar porationelectrode.

FIG. 23 is a plan view of an embodiment employing a plurality ofindependently addressable first poration electrode-second porationelectrode pairs.

FIG. 24A is a plan view of an embodiment employing a planar secondporation electrode and a plurality of independently addressable firstporation electrodes.

FIG. 24B is a cross-sectional view of the embodiment shown in FIG. 24A,taken along line 24B-24B.

DETAILED DESCRIPTION OF THE INVENTION

A preferred embodiment of the invention is now described in detail.Referring to the drawings, like numbers indicate like parts throughoutthe views. As used in the description herein and throughout the claims,the following terms take the meanings explicitly associated herein,unless the context clearly dictates otherwise: the meaning of “a,” “an,”and “the” includes plural reference, the meaning of “in” includes “in”and “on.”

Mass spectrometry systems, methods of use thereof, electrospray systems,methods of use thereof, and methods of fabrication thereof, aredisclosed. The mass spectrometry systems can be operated in a highthroughput (parallel) and/or a multiplexed (individually controlled)mode. The mass spectrometry systems described herein include embodimentsof electrospray systems that are capable of independently forming afluid aerosol (i.e., droplets) and ionizing the molecules present in thefluid. The droplets are formed by producing resonant ultrasonic waves(e.g., acoustical pressure waves) within a reservoir interfaced with astructure having shaped cavities (e.g., acoustic horns) that focus theultrasonic waves and thus amplify the pressure and form a pressuregradient at an ejector nozzle for each shaped cavity. The high pressuregradient close to the ejector nozzle accelerates fluid droplets of sizecomparable to the ejector nozzle diameter (e.g., a few micrometers) outof the ejector nozzle, which are thus controllably generated (e.g.,ejected) during every cycle of the drive signal (e.g., a sinusoidalsignal) after an initial transient. In other words, the droplets areproduced either discretely (e.g., drop-on-demand), or as a continuousjet-based approach.

Decoupling of the droplet generation and the molecular ionizationreduces the energy required to ionize the molecules and also lowers thesample size required, minimizes the dead volume, and improves sampleutilization. In addition, decoupling of the droplet generation and themolecular ionization enables the electrospray system to produce dropletsincluding ionized molecules at low voltages (e.g., about 80 to a fewhundred Volts (V)), in contrast to commonly used electrospray systems(e.g., 1 kV to several kV). In addition, relatively small volumes offluids (e.g., about 100 nanoliters (nL) to a few hundred nL) can be usedin contrast to commonly used electrospray systems (e.g., 3 μL or more).

Embodiments of the electrospray system can be used in a continuous flowonline operation (e.g., continuous loading of samples) and/or indiscrete off-line operation. In discrete off-line operation, embodimentsof the electrospray system can include a disposable nozzle system (e.g.,array of nozzle systems that can include one or more samples andstandards) that can be charged with one or more fluids and inserted intothe electrospray system. The disposable nozzle system can be removed andreplaced with another disposable nozzle system.

Additional embodiments of the electrospray system can be used in a highthroughput electrospray system (e.g., simultaneous use of nozzles)and/or in a multiplexed electrospray system (e.g., using an array ofindividually addressable nozzles or individually addressable groups ofnozzles). Details describing each of these embodiments are described inmore detail below.

FIG. 1 is a schematic of a representative embodiment of a massspectrometry system 10. The mass spectrometry system 10 includes anelectrospray system 12 and a mass spectrometer 14. The electrospraysystem 12 is interfaced with the mass spectrometer 14 so that the fluidsample (e.g., in the form of droplets) is communicated from theelectrospray system 12 to the mass spectrometer system 14 usingelectrostatic lenses and the like under one or more different vacuumpressures. In addition, the electrospray system 12 can be alsointerfaced with a liquid chromatography system, a fluidic system forselective delivery of different samples, and automated fluid chargingsystem such as a pump, for example.

The mass spectrometer 14 can include, but is not limited to, a massanalyzer and an ion detector. The mass analyzer can include, but is notlimited to, a time-of-flight (TOF) mass analyzer, an ion trap massanalyzer (IT-MS), a quadrupole (Q) mass analyzer, a magnetic sector massanalyzer, or an ion cyclotron resonance (ICR) mass analyzer. In someembodiments, because it can be used to separate ions having very highmasses, the mass analyzer is a TOF mass analyzer.

The ion detector is a device for recording the number of ions that aresubjected to an arrival time or position in a mass spectrometry system25, as is known by one skilled in the art. Ion detectors can include,for example, a microchannel plate multiplier detector, an electronmultiplier detector, or a combination thereof. In addition, the massspectrometry system 10 includes vacuum system components and electronicsystem components, as are known by one skilled in the art.

In general, the electrospray system 12 is capable of independentlyforming a fluid aerosol (i.e., droplets) and ionizing the moleculespresent in the fluid. The ionized molecules are then mass analyzed bythe mass spectrometer 14, which can provide information about the typesof molecules present in the fluid sample.

FIG. 2 is an illustration of a cross-section of an embodiment of anelectrospray system 20 a, as shown in FIG. 1. The electrospray system 20a includes, but is not limited to, an array structure 22 includingejector structures 26, a separating layer 28, a reservoir 32, anactuator 42, and an ionization source 44. A fluid can be disposed in thereservoir 32 and in the array 22 of ejector structures 26. Uponactuation of the actuator 42, a resonant ultrasonic wave 52 can beproduced within the reservoir 32 and fluid. The resonant ultrasonic wave52 couples to and transmits through the liquid and is focused by theejector structures 26 to form a pressure gradient 54 within the ejectorstructure 26. The high-pressure gradient 54 accelerates fluid out of theejector structure 26 to produce droplets 56. The cycle of the drivesignal applied to the actuator 42 dictates, at least in part, the rateat which the droplets are discretely produced.

A drop-on-demand ejection can be achieved by modulation of the actuationsignal in time domain. The actuator 42 generating ultrasonic waves canbe excited by a finite duration signal with a number of sinusoidalcycles (a tone burst) at the desired frequency. Since a certain energylevel is reached for droplet ejection, during the initial cycles of thissignal, the standing acoustic wave pattern in the resonant cavity isestablished and the energy level is brought up to the ejectionthreshold. The number of cycles required to achieve the thresholddepends on the amplitude of the signal input to the wave generationdevice and the quality factor of the cavity resonance. After thethreshold is reached, one or more droplets can be ejected in acontrolled manner by reducing the input signal amplitude after thedesired number cycles. This signal can be used repetitively, to eject alarge number of droplets. Another useful feature of this operation is toreduce the thermal effects of the ejection, since the device can cooloff when the actuator 42 is turned off between consecutive ejections.The ejection speed and droplet size can also be controlled by theamplitude and duration of the input signal applied to the actuator 42.

The array structure 22 can include, but is not limited to, an ejectornozzle 24 and an ejector structure 26. In general, the material that thearray structure 22 is made of has substantially higher acousticimpedance as compared to the fluid. The array structure 22 can be madeof materials such as, but not limited to, single crystal silicon (e.g.,oriented in the (100), (010), or (001) direction), metals (e.g.,aluminum, copper, and/or brass), plastics, silicon oxide, siliconenitride, and combinations thereof.

The ejector structure 26 can have a shape such as, but not limited to,conical, pyramidal, or horn-shaped with different cross-sections. Ingeneral, the cross-sectional area is decreasing (e.g., linear,exponential, or some other functional form) from a base of the ejectornozzle 26 (broadest point adjacent the reservoir 32) to the ejectornozzle 24. The cross sections can include, but are not limited to, atriangular cross-section (as depicted in FIG. 2), and exponentiallynarrowing. In an embodiment, the ejector structure 26 is a pyramidalshape.

The ejector structure 26 has acoustic wave focusing properties in orderto establish a highly-localized, pressure maximum substantially close tothe ejector nozzle 24. This results in a large pressure gradient at theejector nozzle 24 since there is effectively an acoustic pressurerelease surface at the ejector nozzle 24. Since the acoustic velocity isrelated to the pressure gradient through Euler's relation, a significantmomentum is transferred to the fluid volume close to the ejector nozzle24 during each cycle of the acoustic wave in the ejector structure 26.When the energy coupled by the acoustic wave in the fluid volume issubstantially larger than the restoring energy due to surface tension,viscous friction, and other sources, the fluid surface is raised fromits equilibrium position. Furthermore, the frequency of the waves shouldbe such that there is enough time for the droplet to break away from thesurface due to instabilities.

The ejector structure 26 has a diameter (at the base) of about 50micrometers to 5 millimeters, 300 micrometers to 1 millimeter, and 600micrometers to 900 micrometers. The distance (height) from the ejectornozzle 24 to the broadest point in the ejector structure 26 is fromabout 20 micrometers to 4 millimeters, 200 micrometers to 1 millimeter,and 400 micrometers to 600 micrometers.

The ejector nozzle 24 size effectively determines the droplet size andthe amount of pressure focusing along with the ejector structure 26geometry (i.e., cavity geometry). The ejector nozzle 24 can be formedusing various micromachining techniques as described below and can havea shape such as, but not limited to, circular, elliptic, rectangular,and rhombic. The ejector nozzle 24 has a diameter of about 50 nanometersto 50 micrometers, 200 nanometers to 30 micrometers, and 1 micrometer to10 micrometers.

In one embodiment all of the ejector nozzles are positioned inline witha mass spectrometer inlet, while in another embodiment only selectejector nozzles (1 or more) are positioned inline with the massspectrometer inlet.

The array structure 22 can include one ejector nozzle 24 (not shown), a(one-dimensional) array of ejector nozzles 24, or a (two dimensional)matrix of parallel arrays of ejector nozzles 24. As shown in FIG. 2, theejector structure 26 can include one ejector nozzle 24 each or include aplurality of ejector nozzles 24 in a single ejector structure 26.

The separating layer 28 is disposed between the array structure 22 andthe actuator 46. The separating layer 28 can be fabricated of a materialsuch as, but not limited to, silicon, metal, and plastic. The separatinglayer 28 is from about 50 micrometers to 5 millimeters in height (i.e.,the distance from the actuator 42 to the array structure 22), from about200 micrometers to 3 millimeters in height, and from about 500micrometers to 1 millimeter in height.

The reservoir 32 is substantially defined by the separating layer 28,the array structure 22, and the actuator 42. In general, the reservoir32 and the ejector structures 26 include the fluid. The reservoir 32 isan open area connected to the open area of the ejector structures 26 sothat fluid flows between both areas. In addition, the reservoir 32 canalso be in fluidic communication (not shown) with a liquidchromatography system or other microfluidic structures capable offlowing fluid into the reservoir 32.

In general, the dimensions of the reservoir 32 and the ejector structure26 can be selected to excite a cavity resonance in the electrospraysystem at a desired frequency. The structures may have cavity resonancesof about 100 kHz to 100 MHz, depending, in part, on fluid type anddimensions and cavity shape, when excited by the actuator 42.

The dimensions of the reservoir 32 are from 100 micrometers to 4centimeters in width, 100 micrometers to 4 centimeters in length, and100 nanometers to 5 centimeters in height. In addition, the dimensionsof the reservoir 32 are from 100 micrometers to 2 centimeters in width,100 micrometers to 2 centimeters in length, and 1 micrometer to 3millimeter in height. Further, the dimensions of the reservoir 32 arefrom 200 micrometers to 1 centimeters in width, 200 micrometers to 1centimeters in length, and 100 micrometers to 2 millimeters in height.

The fluid can include liquids having low ultrasonic attenuation (e.g.,featuring energy loss less than 0.1 dB/cm around 1 MHz operationfrequency). The fluid can be liquids such as, but not limited to, water,methanol, dielectric fluorocarbon fluid, organic solvent, other liquidshaving a low ultrasonic attenuation, and combinations thereof. Thefluids can include one or more molecules that can be solvated andionized. The molecules can include, but are not limited to,polynucleotides, polypeptides, and combinations thereof.

The actuator 42 produces a resonant ultrasonic wave 52 within thereservoir 32 and fluid. As mentioned above, the resonant ultrasonic wave52 couples to and transmits through the liquid and is focused by theejector structures 26 to form a pressure gradient 54 within the ejectorstructure 26. The high-pressure gradient 54 accelerates fluid out of theejector structure 26 to produce droplets. The droplets are produceddiscretely in a drop-on-demand manner. The frequency in which thedroplet are formed is a function of the drive cycle applied to theactuator 42 as well as the fluid, reservoir 32, ejector structure 26,and the ejector nozzle 24.

An alternating voltage is applied (not shown) to the actuator 42 tocause the actuator 42 to produce the resonant ultrasonic wave 52. Theactuator 42 can operate at about 100 kHz to 100 MHz, 500 kHz to 15 MHz,and 800 kHz to 5 MHz. A direct current (DC) bias voltage can also beapplied to the actuator 42 in addition to the alternating voltage. Inembodiments where the actuator 42 is piezoelectric, this bias voltagecan be used to prevent depolarization of the actuator 42 and also togenerate an optimum ambient pressure in the reservoir 32. In embodimentswhere the actuator 42 is electrostatic, the bias voltage is needed forefficient and linear operation of the actuator 42. Operation of theactuator 42 is optimized within these frequency ranges in order to matchthe cavity resonances, and depends on the dimensions of and thematerials used for fabrication of the reservoirs 32 and the arraystructure 22 as well the acoustic properties of the fluids insideejector.

The actuator 42 can include, but is not limited to, a piezoelectricactuator and a capacitive actuator. The piezoelectric actuator and thecapacitive actuator are described in X. C. Jin, I. Ladabaum, F. L.Degertekin, S. Calmes and B. T. Khuri-Yakub, “Fabrication andCharacterization of Surface Micromachined Capacitive UltrasonicImmersion Transducers”, IEEE/ASME Journal of MicroelectromechanicalSystems, 8, pp. 100-114, 1999 and Meacham, J. M., Ejimofor, C., Kumar,S., Degertekin F. L., and Fedorov, A., A micromachined ultrasonicdroplet generator based on liquid horn structure, Rev. Sci. Instrum., 75(5), 1347-1352 (2004), which are incorporated herein by reference.

The dimensions of the actuator 42 depend on the type of actuator used.For embodiments where the actuator 42 is a piezoelectric actuator, thethickness of the actuator 42 is determined, at least in part, by thefrequency of operation and the type of the piezoelectric material. Thethickness of the piezoelectric actuator is chosen such that thethickness of the actuator 42 is about half the wavelength oflongitudinal waves in the piezoelectric material at the frequency ofoperation. Therefore, in case of a piezoelectric actuator, thedimensions of the actuator 42 are from 100 micrometers to 4 centimetersin width, 10 micrometers to 1 centimeter in thickness, and 100micrometers to 4 centimeters in length. In addition, the dimensions ofthe actuator 42 are from 100 micrometers to 2 centimeters in width, 10micrometers to 5 millimeters in thickness, and 100 micrometers to 2centimeters in length. Further, the dimensions of the actuator 42 arefrom 100 micrometers to 1 centimeters in width, 10 micrometers to 2millimeters in thickness, and 100 micrometers to 1 centimeters inlength.

In embodiments where the actuator 42 is an electrostatic actuator, theactuator 42 is built on a wafer made of silicon, glass, quartz, or othersubstrates suitable for microfabrication, where these substratesdetermine the thickness of the actuator 42. Therefore, in case of amicrofabricated electrostatic actuator, the dimensions of the actuator42 are from 100 micrometers to 4 centimeters in width, 10 micrometers to2 millimeter in thickness, and 100 micrometers to 4 centimeters inlength. In addition, the dimensions of the actuator 42 are from 100micrometers to 2 centimeters in width, 10 micrometers to 1 millimeter inthickness, and 100 micrometers to 2 centimeters in length. Further, thedimensions of the actuator 42 are from 100 micrometers to 1 centimetersin width, 10 micrometers to 600 micrometers in thickness, and 100micrometers to 1 centimeter in length.

In the embodiment illustrated in FIG. 2, the ionization source 44 isdisposed on the surface of the actuator 42 adjacent the reservoir 32. Adirect current bias voltage can be applied to the ionization source 44via one or more sources through line 46. The voltage applied to theionization source 44 is substantially lower than that applied incurrently used electrospray systems. The voltage applied to theionization source 44 should be sufficient enough to cause chargeseparation to ionize the molecules present in the fluid. In this regard,the voltage applied to the ionization source 44 should be capable toproduce redox reactions within the fluid. Therefore, the voltage appliedto the ionization source 44 will depend, at least in part, upon thefluid and molecules present in the fluid. The voltage applied to theionization source depends, in part, on the electrochemical redoxpotential of the given sample analyte and is typically from about 0 to±1000 V, ±20 to ±600V, and ±80 to ±300V.

The ionization source 44 can include, but is not limited to, a wireelectrode, a conductive material disposed on the reservoir 32, and anelectrode of the actuator 42, and combinations thereof. The materialthat the wire and/or the conductive material is made of can include, butis not limited to, metal (e.g., copper, gold, and/or platinum),conductive polymers, and combinations thereof. The ionization source 44may cover a small fraction (1%) or an entire surface (100%) of theactuator 42. The ionization source 44 has a thickness of about 1nanometer to 100 micrometers, 10 nanometers to 10 micrometers, and 100nanometers to 1 micrometer.

FIG. 3 is an illustration of a cross-section of another embodiment of anelectrospray system 20 b, as shown in FIG. 1. In this embodiment, asecond ionization source 62 is disposed on portions of the insidesurfaces of ejector structures 26. An electrical potential can beapplied to the second ionization source 62 via one or more sourcesthrough a line 64. As in the embodiment shown in FIG. 2, the secondionization source 62 can be made of similar materials and dimensions.The second ionization source 62 can cover a small fraction (about 1% orjust a tip) or an entire surface (100%) of the nozzle inner surface.This ionization source may not only produce ionization of molecules inthe fluid when operated in DC mode, but also can support formation ofelectrocapillary waves at the fluid interface near the nozzle tip whenoperated in the AC mode in order to facilitate formation the dropletswhose size is even smaller than the nozzle tip opening.

The following fabrication process is not intended to be an exhaustivelist that includes all steps required for fabricating the electrospraysystem 20 b. In addition, the fabrication process is flexible becausethe process steps may be performed in a different order than the orderillustrated in FIGS. 4A through 4J.

FIGS. 4A through 4J are illustrations of cross-sections of arepresentative embodiment of a method of forming the electrospray systemshown in FIG. 3. FIG. 4A illustrates an array substrate 72 having afirst masking layer 74 disposed thereon and patterned usingphotolithographic techniques. The first masking layer 74 can be formedof materials such as, but not limited to, a silicon nitride mask(Si₃N₄). The first mask layer 74 can be formed using techniques such as,but not limited to, plasma enhanced chemical vapor deposition, lowpressure chemical vapor deposition, and combinations thereof. Thepatterning of the first masking layer 74 is done using standardphotolithography techniques.

FIG. 4B illustrates the array substrate 72 after being etched to formthe array structure 22 having ejector structures 26 formed in areaswhere the mask 74 was not disposed. The etching of the array substrate72 to form the ejector structures 26. The etching technique can include,but is not limited to, a potassium hydroxide (KOH) anisotropic etch,reactive ion etching (RIE), and inductively coupled plasma etch (ICP),and focused ion beam (FIB) machining. It should also be noted that thearray substrate 72 can be formed via stamping, molding, or othermanufacturing technique.

An example of etching includes, but is not limited to, the formation ofa pyramidal ejector structure having internal wall angles of about54.74° using anisotropic KOH etch of a single crystal silicon wafer fromthe (100) surface. The KOH solution etches the exposed (100) planes morerapidly than the (111) planes to form the pyramidal ejector structuresuch as described in Madou, M. J. (2002). Fundamentals ofMicrofabrication. Boca Raton, Fla., CRC Press.

FIG. 4C illustrates the removal of the first masking layer 74 using areactive ion etching (RIE) process or similar process, if necessary,while FIG. 4D illustrates the addition of a second masking 76. Thesecond masking layer 76 can be formed of materials such as, but notlimited to, a photoresist mask, a silicon nitride (hard) mask (Si₃N₄),and a silicon oxide (hard) mask (SiO₂) which is patterned usingphotolithography techniques. The second masking layer 76 can be formedusing techniques such as, but not limited to photolithography etching,inductively coupled plasma (ICP) etching, and reactive ion etching(RIE), and combinations thereof.

FIG. 4E illustrates the etching of the second mask layer 76 to form theejector nozzle 24 in the array substrate 22. The etching technique caninclude, but is not limited to, photolithography etching, inductivelycoupled plasma (ICP) etching, and reactive ion etching (RIE).Alternatively, depending on the size and geometry, the ejector nozzles24 a and 24 b can be cut from the wafer, using a dicing saw or othersimilar device. Also, the ejector nozzles 24 a and 24 b can be machinedusing focused ion beam (FIB), and laser or electron beam (E-beam)drilling as opposed to using the second mask layer 76.

FIG. 4F illustrates the removal of the second mask layer 76 using areactive ion etching (RIE) process or similar process. FIG. 4Gillustrates the deposition of the second ionization source 62 on theinside wall of the ejector structure 26. The deposition techniques caninclude, but is not limited to, evaporation, sputtering, chemical vapordeposition (CVD), and electroplating.

FIG. 4H illustrates the placement of the separating layer 28 on portionsof the array structure 22 to form the lower portion 82 of theelectrospray system 20 b. The separating layer 28 can be made separatelyby etching silicon, machining of the metal, or stamping the polymer.Once fabricated, this separating layer 28 can be bonded to the arraystructure 22 using a polyimide layer (such as Kapton™ or other bondingmaterial). This dry film can be laminated and patterned using lasermicromachining or photolithography techniques. The separating layer 28can then be affixed/bonded to the piezoelectric transducer to form theoperational device. Alternatively, the separating layer 28 is bonded tothe upper portion 84 using a polyimide layer, for example. Then theseparating layer 28 is bonded to the array structure 22.

FIG. 4I illustrates the lower portion 82 of the electrospray system 20 band the upper portion 84 of the electrospray system 20 b, while FIG. 4Jillustrates the formation of the electrospray system 20 b by joining(e.g., bonding and/or adhering) the lower portion 82 and the upperportion 84. It should be noted that the lower portion 82 could beproduced separately and be used as a disposable cartridge that isreplaced regularly on the electrospray system 20 b, while the upperportion 84 is reused. In another embodiment not shown, the lower portion82 does not include the separating layer 28 and the separating layer 28is disposed on the upper portion 84, so that the upper portion 84 withthe separating layer 28 disposed thereon is reused. In still anotherembodiment, the separating layer 28 can be removed separately fromeither the upper portion 84 and the lower portion 82.

FIG. 5 is an illustration of a cross-section of another embodiment of anelectrospray system 12, as shown in FIG. 1. In this embodiment, theelectrospray system 100 includes a first reservoir 32 a and a secondreservoir 32 b. In addition, the first reservoir 32 a and the secondreservoir 32 b each are adjacent a first actuator 42 a and a secondactuator 42 b, respectively. Furthermore, the first reservoir 32 a andthe second reservoir 32 b each are adjacent a first ejector structure 24a and a second ejector structure 24 b, respectively.

The first reservoir 32 a and the second reservoir 32 b are separated bya center separating layer 28 c. The first reservoir 32 a is bound by thefirst separating layer 28 a, the center separating layer 28 c, the firstactuator 42 a, and the first ejector structure 26 a. The secondreservoir 32 b is bound by the second separating layer 28 b, the centerseparating layer 28 c, the second actuator 42 b, and the second ejectorstructure 26 b. The same or a different fluid can be disposed in thefirst reservoir 32 a and the second reservoir 32 b, chosen to match theacoustic properties of the sample loaded in the cavity of the ejectorstructures 26 a and 26 b, respectively. This configuration allows one togenerate electrosprays of different fluids by simply electronicallychoosing the first actuator 42 a, or the second actuator 42 b. Thenumber of the reservoirs can be increased by replicating this structurein the lateral dimension.

FIG. 6 is an illustration of a cross-section of another embodiment of anelectrospray system 12, as shown in FIG. 1. Similar to the electrospraysystem 100 shown in FIG. 5, the electrospray system 120 shown in FIG. 6includes a first reservoir 32 a and a second reservoir 32 b. The firstreservoir 32 a is bound by the first separating layer 28 a, the centerseparating layer 28 c, the first actuator 42 a, and the first ejectorstructure 22 a. The first reservoir 32 a includes a gas bubble (notshown). The second reservoir 32 b is bound by the second separatinglayer 28 b, the center separating layer 32 c, a second actuator 42 b,and the second ejector structure 22 b. The second reservoir 32 bincludes a fluid bubble 208.

In addition, as shown in FIG. 7, the electrospray system 120 includes afirst separating structure 132 a and a second separating structure 132b, each disposed on top of the first ejection structure 26 a and thesecond ejection structure 26 b, respectively, separating the firstreservoir 32 a and the second reservoir 32 b from the first arraystructure 22 a and second array structure 22 b, respectively. Asdemonstrated later with respect to FIGS. 8A through 8K, the first arraystructure 22 a and second array structure 22 b are filled with a firstfluid 134 a and a second fluid 134 b, respectively, and then the firstseparating structure 132 a and the second separating structure 132 b aredisposed on top of the first ejection structure 26 a and the secondejection structure 26 b. It should be noted that the electrospray system120 does not include a first ionization source 44 a and 44 b since thefirst actuator 42 a and the second actuator 42 b are separated from thefirst fluid 134 a and the second fluid 134 b. This allows forindividually addressable ionization sources, whose potential can beindividually controlled.

The first separating structure 132 a and the second separating structure132 b can be one structure or two distinct structures, which show littleimpedance to propagation of acoustic waves at the operation frequency ofthe actuators 42 a and 42 b. The first separating structure 132 a andthe second separating structure 132 b can be made of materials such as,but not limited to polyimide layer (such as Kapton™), pyrolene, andother suitable materials. The first separating structure 132 a and thesecond separating structure 132 b can have a thickness of about 1micrometers to 200 micrometers. The length and width of the firstseparating structure 132 a and the second separating structure 132 bwill depend upon the dimensions of the first array structure 22 a andsecond array structure 22 b.

The first fluid 134 a can be ejected out of the first ejection structure26 a by controllably positioning the fluid bubble (not shown)substantially between the first separating structure 132 a and the firstactuator 42 a to fill in the reservoir 32 a. Likewise, the second fluid134 b can be ejected out of the second ejection structure 26 b bycontrollably positioning the fluid bubble 208 substantially between thesecond separating structure 132 b and the second actuator 42 b to fillin the reservoir 32 b.

The ejection of the first fluid 134 a and second fluid 134 b can becontrolled in at least two ways for the electrospray system 120 shown inFIG. 6. First, the first actuator 42 a and the second actuator 42 b canbe individually activated to cause ejection of the first fluid 134 a andthe second fluid 134 b if the fluid bubble 208 is properly positioned.Second, a gas bubble (not shown) can be positioned substantially betweenthe first separating structure 132 a and the first actuator 42 a and/orthe second separating structure 132 b and the second actuator 42 b.Since the gas bubble does not effectively couple to and transmit theultrasonic pressure wave, the first fluid 134 a and the second fluid 134b will not be ejected, even if the first actuator 42 a and/or the secondactuator 42 b are activated. The process for selectively ejecting fluidfrom one or more ejector structures is described in further detail inFIGS. 9A though 9D and 10A through 10F.

FIG. 7 is an illustration of a cross-section of another embodiment of anelectrospray system 12, as shown in FIG. 1. In contrast to theelectrospray system 120 in FIG. 6, the electrospray system 150 shown inFIG. 7 includes only a single actuator 42 in communication with thefirst reservoir 32 a and the second reservoir 32 b. As in theelectrospray system 120 in FIG. 6, the first fluid 134 a can be ejectedout of the first ejection structure 26 a by controllably positioning thefluid bubble (not shown) substantially between the first separatingstructure 132 a and the first actuator 42 a to fill in the reservoir 32a. Likewise, the second fluid 134 b can be ejected out of the secondejection structure 26 b by controllably positioning the fluid bubble 208substantially between the second separating structure 132 b and thesecond actuator 42 b to fill in the reservoir 32 b.

In addition, the first fluid 134 a can not be ejected out of the firstejection structure 26 a when the gas bubble (not shown) is positionedsubstantially between the first separating structure 132 a and the firstactuator 42 a to fill in the reservoir 32 a. Likewise, the second fluid134 b can not be ejected out of the second ejection structure 26 b whenthe gas bubble (not shown) is positioned substantially between thesecond separating structure 132 b and the second actuator 42 b to fillin the reservoir 32 b.

Therefore, upon actuation of the actuator 42 and positioning of thefluid bubble 208 and the gas bubble, the ejection of the first fluid 134a and the second fluid 134 b can be selectively controlled. For example,in the configuration in FIG. 7, actuation of the actuator 42 causes thesecond fluid 134 b to be ejected, while the first fluid 134 a is notejected. The process for selectively ejecting fluid from one or moreejector structures is described in further detail in FIGS. 9A though 9Cand 10A through 10E.

The following fabrication process is not intended to be an exhaustivelist that includes all steps required for fabricating the electrospraysystem 150. In addition, the fabrication process is flexible because theprocess steps may be performed in a different order than the orderillustrated in FIGS. 8A through 8K.

FIGS. 8A through 8K are illustrations of cross-sections of arepresentative embodiment of a method of forming the electrospray systemshown in FIG. 7. FIG. 8A illustrates an array substrate 72 having afirst masking layer 144 disposed thereon. The first masking layer 144can be formed of materials such as, but not limited to, a siliconnitride mask (Si₃N₄), silicon oxide (SiO₂) and patterned using standardphotolithography techniques. The first mask 144 can be disposed usingtechniques such as, but not limited to, inductively coupled plasma (ICP)etch, reactive ion etch (RIE), or wet etching.

FIG. 8B illustrates the array substrate 72 after being etched to formthe first array structure 22 a and the second array structure 22 bhaving the first ejector structures 26 a and the second ejectorstructure 26 b formed in areas where the mask 144 was not disposed. Theetching of the array substrate 72 to form the first ejector structures26 a and the second ejector structure 26 b). The etching technique caninclude, but is not limited to, a potassium hydroxide (KOH) anisotropicetch of (100) single crystal silicon and laser micro-machining.

FIG. 8C illustrates the removal of the first mask 144 using a reactiveion etching (RIE) process or similar process, and FIG. 8D illustratesthe addition of a second masking layer 152. The second mask 152 can beformed of materials such as, but not limited to, a silicon nitride mask(Si₃N₄), a silicon oxide mask (SiO₂), or a photoresist.

FIG. 8E illustrates the etching of the second mask 152 to form theejector nozzles 24 a and 24 b for the first ejector structure 26 a andthe second ejector structure 26 b, respectively. The etching techniquecan include, but is not limited to, photolithography etching,inductively coupled plasma (ICP) etching, reactive ion etching (RIE),and wet chemical etching. Alternatively, depending on the size andgeometry, the ejector nozzles 24 a and 24 b may be cut from the wafer,using a dicing saw or other similar device, and can be machined usingfocused ion beam (FIB), and laser or electron beam (E-beam) drilling, asopposed to using the second mask 152. FIG. 8F illustrates the removal ofthe second mask 152 using a reactive ion etching (RIE) process orsimilar process.

FIG. 8G illustrates the deposition of the second ionization source 62 aand 62 b on the inside wall of the first ejector structure 26 a and thesecond ejector structure 26 b, respectively. The deposition techniquescan include, but are not limited to, evaporation, sputtering, chemicalvapor deposition, and electroplating.

FIG. 8H illustrates the formation of the first separating structure 132a and the second separating structure 132 b (these structures can be thesame or be two distinct structures). In addition, an ejector nozzlesealing structure 136 is disposed on top of the ejector nozzles 24 a and24 b of the first ejector structure 26 a and second ejector structure 26b. The ejector nozzle sealing structure 136 can be made of materialssuch as, but not limited to, polyimide layer (such as Kapton) or someother inert layer such as parylene film.

Prior to the formation of the first separating structure 132 a and thesecond separating structure 132 b, the first ejector structure 26 a andsecond ejector structure 26 b are filled with a first fluid 134 a and asecond fluid 134 b. The first fluid 134 a and the second fluid 134 b canbe the same fluid or different fluids.

FIG. 8I illustrates the placement of the first separating layer 28 a,the second separating layer 28 b, and a center separating layer 28 d onportions of the first array structure 22 a and the second arraystructure 22 b to form the lower portion 152 of the electrospray system150. The first separating layer 28 a, the second separating layer 28 b,and a center separating layer 28 d can each be made separately byetching silicon or simple machining of the metal or stamping thepolymer. Once fabricated, the first separating layer 28 a, the secondseparating layer 28 b, and a center separating layer 28 d each can bebonded to the nozzle array using a polyimide layer (such as Kapton).This dry film can be laminated and patterned using laser micro-machiningor photolithography techniques. This spacer layer can then beaffixed/bonded to the piezoelectric transducer to form the operationaldevice.

It should be noted that the first separating layer 28 a, the secondseparating layer 28 b, and a center separating layer 28 d can bedisposed on portions of the first array structure 22 a and the secondarray structure 22 b prior to the formation of the first separatingstructure 132 a and the second separating structure 132 b and/or theejector nozzle sealing structure 136. In addition, the first fluid 134 aand the second fluid 134 b can be disposed in the first ejectorstructure 26 a and second ejector structure 26 b after the firstseparating layer 28 a, the second separating layer 28 b, and the centerseparating layer 28 d are formed.

In this regard, a structure including the first ejector structure 26 aand the second ejector structure 26 b and the first separating layer 28a, the second separating layer 28 b, and the center separating layer 28d can be produced. Then in a separate process, the ejector nozzlesealing structure 136 can be positioned adjacent the first ejectornozzle 24 a and the second ejector nozzle 24 b, respectively.Subsequently, the first fluid 134 a and the second fluid 134 b can bedispensed into the first ejector structure 26 a and second ejectorstructure 26 b, respectively. Lastly, the first separating structure 132a and the second separating structure 132 b can be disposed on the topof the first ejector nozzle 24 a and the second ejector nozzle 24 b,respectively.

In another embodiment not shown, the lower portion 152 does not includethe first separating layer 28 a, the second separating layer 28 b, andthe center separating layer 28 d. The first separating layer 28 a, thesecond separating layer 28 b, and the center separating layer 28 d aredisposed on the upper portion 154. Therefore, the upper portion 154 withthe first separating layer 28 a, the second separating layer 28 b, andthe center separating layer 28 d disposed thereon can be reused. Instill another embodiment, the first separating layer 28 a, the secondseparating layer 28 b, and the center separating layer 28 d can beremoved separately from either the upper portion 154 or the lowerportion 152.

FIG. 8J illustrates the lower portion 152 of the electrospray system 150and the upper portion 154 of the electrospray system 150, and FIG. 8Killustrates the formation of the electrospray system 150 by joining(e.g., bonding and/or adhering) the lower portion 152 and the upperportion 154. It should be noted that the lower portion 152 could beproduced separately and be used as a disposable cartridge that isreplaced regularly on the electrospray system 150, while the upperportion 154 is reused.

FIGS. 9A through 9D are illustrations of top views of representativeembodiments of an electrospray system 200. FIG. 9B illustrates a fluidbubble in one section of the electrospray system 200, while FIG. 9Cillustrates a fluid bubble in the other section of the electrospraysystem 200. The electrospray system 200 has a single actuator (notshown) positioned in communication with a first reservoir 202 a and asecond reservoir 202 b. The first reservoir 202 a and the secondreservoir 202 b are separated from each other by a separating layer 206.The first reservoir 202 a and the second reservoir 202 b are separatedfrom the array structure (not shown) having a first ejector structure204 a and a second ejector structure 204 b by a first separatingstructure and a second separating structure (not shown). The firstejector structure 204 a and the second ejector structure 204 b eachcontain a fluid within their respective cavities.

FIG. 9A illustrates the electrospray system 200 in a state where onlygas bubbles (not shown) are positioned within the first reservoir 202 aand the second reservoir 202 b. As mentioned above, a gas bubble doesnot effectively couple to and transmit the ultrasonic pressure wave, soupon actuation of the actuator substantially no fluid is ejected fromthe first ejector structure 204 a and the second ejector structure 204b.

FIG. 9B illustrates an acoustically responsive fluid bubble 208 in thesecond reservoir 202 b of the electrospray system 200. Since the fluidbubble 208 can substantially couple to and transmit the ultrasonicpressure wave, actuation of the actuator causes the fluid within thesecond ejector structure 204 b to be ejected through the ejectorsnozzles of the second ejector structure 204 b, but substantially nofluid is ejected from the first ejector structure 204 a since the gasbubble does not effectively couple to and transmit the ultrasonicpressure wave produced by the actuator.

FIG. 9C illustrates an acoustically responsive fluid bubble 208 in thefirst reservoir 202 a of the electrospray system 200. Since the fluidbubble 208 can substantially couple to and transmit the ultrasonicpressure wave, actuation of the actuator causes the fluid within thefirst ejector structure 204 a to be ejected through the ejectors nozzlesof the first ejector structure 204 a, but substantially no fluid isejected from the second ejector structure 204 b since the gas bubbledoes not effectively couple to and transmit the ultrasonic pressure waveproduced by the actuator.

FIG. 9D illustrates acoustically responsive fluid bubbles 208 in thefirst reservoir 202 a and the second reservoir 202 b of the electrospraysystem 200. Since the fluid bubble 208 can substantially couple to andtransmit the ultrasonic pressure wave, actuation of the actuator causesthe fluid within the first ejector structure 204 a and the secondejector structure 204 b to be ejected through the ejectors nozzles ofthe first ejector structure 204 a and the second ejector structure 204b.

FIGS. 10A through 10F are illustrations of top views of representativeembodiments of an electrospray system 220 that may be used in amultiplexing format and/or parallel analysis. FIGS. 10B through 10Eillustrate an acoustically responsive fluid bubble 208 being positionedfrom one section of the electrospray system 220 to another. Theelectrospray system 220 has a single actuator (not shown) positioned incommunication with a first reservoir 222 a, a second reservoir 222 b, athird reservoir 222 c, and a fourth reservoir 222 d. The first reservoir222 a, the second reservoir 222 b, the third reservoir 222 c, and thefourth reservoir 222 d are separated from each other by a firstseparating layer 226 a and a second separating layer 226 b. The firstreservoir 222 a, the second reservoir 222 b, the third reservoir 222 c,and the fourth reservoir 222 d are separated from the array structure(not shown) having a first ejector structure 224 a, a second ejectorstructure 224 b, a third ejector structure 224 c, and a fourth ejectorstructure 224 d, by a first separating structure, a second separatingstructure, a third separating structure, and a fourth separatingstructure (not shown). The first reservoir 222 a, the second reservoir222 b, the third reservoir 222 c, and the fourth reservoir 222 d, eachcontain a fluid within their respective cavities.

FIG. 10A illustrates the electrospray system 220 in a state where onlygas bubbles (not shown) are positioned within the first reservoir 222 a,the second reservoir 222 b, the third reservoir 222 c, and the fourthreservoir 222 d. As mentioned above, a gas bubble does not effectivelycouple to and transmit the ultrasonic pressure wave. Thus, uponactuation of the actuators substantially no fluid is ejected from thefirst ejector structure 224 a, the second ejector structure 224 b, thethird ejector structure 224 c, and the fourth ejector structure 224 d.

Similar to FIGS. 9A through 9D, an acoustically responsive fluid bubble208 is controllably moved from the first reservoir 222 a to the fourthreservoir 224 c in a stepwise manner in FIGS. 10B through 10E. Since thefluid bubble 208 can substantially couple to and transmit the ultrasonicpressure wave, actuation of the actuator causes the fluid within theejector structure having the fluid bubble disposed in the correspondingreservoir to be ejected through the ejectors nozzles of the that ejectorstructure. However, substantially no fluid is ejected from the otherejector structures since the gas bubble does not effectively couple toand transmit the ultrasonic pressure wave produced by the actuator.

FIG. 10F illustrates an acoustically responsive fluid bubble 208 in thefirst reservoir 222 a and the fourth reservoir 224 c. Since the fluidbubble 208 can substantially couple to and transmit the ultrasonicpressure wave, actuation of the actuator causes the fluid within firstejector structure 224 a and the fourth ejector structure 224 d to beejected through the ejectors nozzles of the each ejector structure. Inother embodiments, the fluid bubble 208 can be positioned in one or moreof the reservoirs so that one or more fluids within the ejectorstructures can be ejected simultaneously.

While embodiments of electrospray system are described in connectionwith Examples 1 and 2 and the corresponding text and figures, there isno intent to limit embodiments of the electrospray system to thesedescriptions. On the contrary, the intent is to cover all alternatives,modifications, and equivalents included within the spirit and scope ofembodiments of the present disclosure.

EXAMPLE 1 On-Demand Droplet Formation and Ejection Using MicromachinedUltrasonic Atomizer

While embodiments of electrospray system are described in connectionwith examples 1 and 2 and the corresponding text and figures, there isno intent to limit embodiments of the electrospray system to thesedescriptions. On the contrary, the intent is to cover all alternatives,modifications, and equivalents included within the spirit and scope ofembodiments of the present disclosure. An exemplary embodiment of arepresentative electrospray system has been developed and tested on amass spectrometer (MS). As shown in FIG. 11, it includes of apiezoelectric transducer, a fluid reservoir, and a silicon cover platecontaining the micromachined ejector nozzles, similar to the design inFIG. 1. A PZT-8 ceramic is selected for the piezoelectric transducer.The device generates droplets by utilizing cavity resonances in theabout 1 to 5 MHz range, along with the acoustic wave focusing propertiesof liquid horns formed by a silicon wet etching process. At resonance, astanding acoustic wave is formed in the fluid reservoir with the peakpressure gradient occurring at the tip of the nozzle leading to dropletejection. Finite element analysis using ANSYS (2003) not only confirmsthe acoustic wave focusing by the horn structure shown in FIG. 11, butalso accurately predicts the resonant frequencies at which the deviceprovides stable droplet ejection.

Although a number of horn shapes are capable of focusing acoustic waves,a pyramidal shape was selected as it can be readily fabricated via, forexample, a single step potassium hydroxide (KOH) wet etch of (100)oriented silicon. As shown in FIG. 12, when square patterns are openedin a mask layer material, such as silicon nitride (FIG. 12, steps 2 and3), deposited on the surface of a (100) oriented silicon wafer, and theedges are aligned to the <110> directions, the KOH solution etches theexposed (100) planes more rapidly than the (111) planes yielding apyramid shaped horn (FIG. 12, step 4) making a 54.74° angle with theplane of the wafer. The sizes of the square features representing thebase of the pyramid are designed so that the tip of these focusingpyramidal horns terminate within about 1 to 20 μm of the oppositesurface of the ejector plate.

As the last step of the process, the nozzles of the desired diameter(about 3 to 5 μm in this embodiment) are formed by exemplary dry etchingthe remaining silicon from the opposite side in inductively coupledplasma (ICP) using a patterned silicon oxide layer as the hard mask(FIG. 12, steps 6 and 7). As shown in the Scanning Electron Micrographs(SEMs) in FIGS. 13A and 13B, this simple exemplary process, with onlytwo masks and two etching steps, has been used to fabricate hundreds ofpyramidal horns with nozzles on a single silicon wafer.

FIGS. 14A through 14C illustrate the device in operation, where theclouds of generated aerosol are emanating from the device. FIGS. 14B and14C show enhanced stroboscobic images of about 8 μm and about 5 μmdiameter water droplets ejected from a single nozzle on differentwafers, at a frequency of about 1.4 MHz and about 916 kHz, respectively.By making the nozzles even smaller or exploiting the instabilities ofthe liquid interface during droplet formation (e.g., by promotionformation of electrocapillary waves at the fluid interface), it may bepossible to produce even smaller, sub-micron droplets using this dropletgeneration technology.

EXAMPLE 2 Electrospray Generation of Protein Ions at Low AppliedVoltages and MS Analysis

Protein ions suitable for high sensitivity mass spectrometric analysiswith an ionization voltage below 300 V (rather than kilovolts requiredby the conventional nanospray sources) have been produced usingembodiments of the electrospray system. FIG. 15 illustrates a schematicof the experimental setup in which an electrode of the piezoelectrictransducer is also used for electrochemical charging of the fluid byapplying DC bias voltage in addition to the AC signal used for soundwaves generation. FIG. 16 shows a strong peak of the 609 Da molecularweight compound (with signal-to-noise ratios of 3 or better) obtained inMS analysis of the mixture containing a standard low molecular weighttest peptide, such as reserpine (MW=609 Da, CAS#50-55-5), ionized usingthe embodiment of the electrospray system.

One embodiment of the invention may be used in cellular manipulation,such as: lysis (disruption of a cell membrane and removal of materialfrom the cell), poration (opening pores in a cell membrane to enablematerial transfer to and from the cell), transfection (moving materialinto cells through the cell membrane) and sorting. As shown in FIG. 17A,a cellular manipulation embodiment includes a substrate 310 (such as asilicon wafer) defining a plurality of tapering passages 312 (such aspyramidal or frusto-conical passages) that terminate in openings 314passing through the substrate 310. Each tapering passage 312 includes afirst poration electrode 320, electrically coupled to a first electricalcontact 324, and a spaced-apart second poration electrode 322,electrically coupled to a second electrical contact 326 so that when apotential is applied between the first contact 324 and the secondcontact 326, an electric field will form between the first porationelectrode 320 and the second poration electrode 322. A bias voltage 328may also be applied to the substrate 310. Also, an oscillator may beused to drive the poration electrodes, thereby inducing an oscillatingelectric field.

An actuator 330 a and 330 b is spaced apart from the substrate 310 so asto form a cavity 306 therebetween. The actuator 330 a and 330 b aredriven by an oscillator 338 to cause generation of an acoustic wave. Ifa fluid is placed in the cavity 306, then the acoustic wave will befocused by the tapering passages 312 onto the fluid. The spacing of theoscillator 338 from the substrate 310 and selection of the frequency ofoscillation will determine the nature of the acoustic wave, and thesevariables may be tuned so as to generate a standing acoustic wave in thetapering passages 312. The acoustic wave may be focused by the passage312 so that it has a predetermined compression geometry relative to thepassage. Such a wave has a highly predictable pressure gradient thatensures that any cells placed in the tapering passages will be subjectto a predetermined pressure at any given point along the taperingpassage 312. Typically, the cells are suspended in a liquid placed intothe cavity 306. The acoustic wave can then induce sonoporation of cellsand can drive the cells through the openings 314 as ejected material304. Thus, this embodiment may act as an electrostatic gun fortransporting cellular material.

The actuator 330 a and 330 b, which can include an ultrasonictransducer, can include a layer of piezoelectric material 334 disposedbetween a first transducer electrode 332, which may be biased with abias voltage 340, and an opposite second transducer electrode 336. Theactuator 330 a and 330 b is oriented so that when a potential is appliedbetween the first transducer electrode 332 and the second transducerelectrode 336 (such as with the oscillator 338), the layer ofpiezoelectric material 334 expands or contracts, thereby generating anacoustic wave.

It is also possible to employ a capacitive transducer, that wouldinclude the first transducer electrode 332 and the second transducerelectrode 336, but have only an air gap therebetween. When a potentialis applied between the first transducer electrode 332 and the secondtransducer electrode 336, the second transducer electrode 336 movesrelative to the first transducer electrode 332, thereby generating awave.

When a potential is applied between the first poration electrode 320 andthe second poration electrode 322, an electric field is generated. Theelectric field can cause electroporation of the cells. The combinationof the electroporation and sonoporation can give rise to highlypredictable poration of the cells. As the cell passes through theopening 314 the cell membrane allows the substance to pass therethrough.If a biologic material or a chemical composition (e.g., DNA, RNA, othergenetic material, a pharmaceutical, a nano-particle, a dye, an imagingcomposition etc.) is placed in the liquid with the cells, then some ofthe material will pass into the cells as a result of the poration of thecells.

Likewise, if the electric field and acoustic wave have sufficient energygradients, then highly predictable lysis can occur with the cells. Thismay be used to extract cellular material (e.g., DNA, RNA, genes,organelles, etc.) from the cells.

This embodiment may also be used in sorting cells by size. If the sizeof the openings 314 is such that only those cells smaller that a givensize will pass through the openings, then the lager cells will staybehind.

In the embodiment shown in FIG. 17B, first poration electrode may beco-incidental with the second transducer electrode 336 (and biased withvoltage 352) and the second poration electrode 350 may be disposedadjacent to the second side of the substrate. Also, in one example, adopant may also be added to the substrate 310 to allow it to act as aporation electrode.

A plan view of one embodiment is shown in FIG. 18A, showing thesubstrate 310 and the openings 314. An opposite view is shown in FIG.18B, showing the substrate 310, the tapering passages 312 and theopenings 314. A cross section of this embodiment is shown in FIG. 18C.FIG. 19 shows a micrograph 356 of one of the tapering passages.

One embodiment, as shown in FIGS. 20A and 20B, may be used to processdifferent analytes simultaneously. In this embodiment, the transducer330 (which is shown as a unitary transducer in this example) may bedetachable from the substrate 310 and the different analytes are placedinto the passages 312 using a pipette 370. In the example shown a firstanalyte 360 is placed in several of the passages 312 and a secondanalyte 362 is placed in the remaining passages 312. The transducer 330is replaced, as shown in FIG. 20B and operation continues as describedabove. In this example, the transducer 330 may be placed directlyadjacent to the substrate 310, without an intervening cavity. In thisexample the first analyte 360 could include a first type of cell withthe second analyte 362 could include a second type of cell. Also,different additives (such as different types of dye) may distinguishbetween the first analyte 360 and the second analyte 362. More than twodifferent types of analyte may be analyzed simultaneously with thisembodiment.

The invention may be used to manipulate cells continuously, as shown inFIG. 21, through use of a fluid pump 374 a and 374 b. In such anembodiment, as the cellular suspension is driven out of the passages312, it is replaced by new fluid from the fluid pump 374 a and 374 b.Placing a wall 370 between a first portion of the passages and a secondportion of the passages allows for analysis of different fluids (thefirst from a first fluid pump 374 a and the second from a second fluidpump 374 b) and with different energies (for example, using a firstacoustic wave energy from the first acoustic transducer 330 a and asecond acoustic wave energy from the second acoustic transducer 330 b).

As shown in FIG. 22, the second poration electrode 380 might be disposedon the top surface of the substrate 310. Also, as shown in FIG. 23, thefirst poration electrode 390 may include a plurality of conductivestrips placed on the substrate 310 along each row of openings 314. Thesecond poration electrode 392 may also include a plurality of conductivestrips placed on the substrate 310 along each row of openings 314. Thisway, each strip might be a separately addressable sub-electrode to allowfor the application of a different potential (ΔV₁-ΔV₆) for each porationsub-electrode pair.

Another way to accomplish the application of different electrical fieldsbeing applied to different passages 312 is shown in FIGS. 24A and 24B.In this embodiment, the second poration electrode 380 is applied to thesubstrate, a layer of an insulator layer 398 is applied onto the firstporation electrode 380 and then a first poration electrode layer isapplied to the insulator layer 398. The first poration electrode layeris patterned (e.g., through etching) to create a plurality ofrow-specific addressable first poration sub-electrodes 396.

One experimental embodiment includes an electrostatic gun for injectingDNA into cells and for sorting cells according to size. The embodimentincludes an array of conical horn structures or pyramidal passages. Eachhorn structure includes a pair of spaced-apart electroporationelectrodes that apply a potential across cell membranes. Each hornstructure opens to an orifice that has a diameter corresponding to atarget cell size. Behind each horn structure is a piezoelectrictransducer that provides an ultrasonic pressure wave to transportanalyte and enhance poration (via sonoporation).

The device provides on-demand DNA delivery in or out of the cell viacombination (or possibly individual action) of ultrasonic and electricalporation or lysis, respectively, of the cell membrane. In addition toporation and lysing functionality, the device also includes thecapability for inline size selective cell sorting (via control of theejector nozzle size) prior to poration/lysis. It also enables transportof modified cell/DNA to final destination as a post-poration/lysis stepfor further processing. The device can operate in both high-throughputand multiplexed mode in the microarray format.

The electro-sonic DNA gun is designed to work in an array format, so itcan operate in both high throughput mode, and also in the multiplexedmode if the array is divided into individually controlled compartments.Each compartment is loaded with an analyte that contains a buffersolution, suspension of biological cells, and a DNA transport that onedesires to inject into the cells. Different analytes may be loaded intodifferent compartments. The horn nozzle structures of the analyte loadedchambers efficiently focus acoustic waves generated by driving thepiezoelectric transducer at one of the resonant frequencies of the fluidcavities, leading to establishment of a significant pressure gradientnear the tip of the nozzles. This pressure gradient at the nozzle tipserves two important functions: (1) it allows to eject on-demanddroplets of the analyte from the device into the cell; and (2) it allowsstrong and limited duration application of mechanical force to the cellmembrane as it passes through the nozzle neck during the ejection,leading to either membrane poration and injection of DNA and RNA fromthe solution into the cell through open pores or cell membrane rupture(lysis) and release of the cell content into the buffer solution. Inboth cases, a drop, containing buffer solution together with either acell injected with DNA or DNA released from the lysed cell is beingejected and could be delivered to the specific location or destinationpoint for further processing. Efficient sonoporation occurs whenamplitude of the acoustic pressure pulse applied to the cell membrane isbetween 1 and 100 kPa (in access of the DC hydrostatic pressure) and thepulse duration in the range of 0.1 and 10 μs—these operating parametersare readily realized in operation of the electro-sonic DNA gun byvarying an amplitude and modulating frequency of the piezoelectrictransducer driving frequency.

Simultaneously with acoustic pumping, cell poration (for DNA injection)or rupture (for DNA extraction) can be accomplished via application ofAC or DC electric field to the electroporation electrodes depositedwithin the nozzles of the device fluid chambers. Because of the closeproximity of electrodes (separation distance ranging from 10 μm to100ths of a micrometer) fairly small voltages of the order of 1 to 10Volts are needed to achieve electric field strengths of 1 kV/cm requiredfor electroporation. Typical electric signal pulse length required forelectroporation is between 100 ns and 100 μs, and is readily realized bythe disclosed electro-sonic DNA gun when operated in either MHzfrequency range or in kHz domain by using time-domain amplitudemodulation of the driving signal. Finally, since the size of thedroplets that can be ejected from the device is dictated by the size ofthe nozzle orifice, it allows for cell separation and sorting throughsize exclusion immediately after DNA injection and extraction. The sizeof the realized nozzles (3 to 30 μm) corresponds well with the size ofeukaryotic animal cells (typically 5-30 μm; for example, red blood cellsare ˜7-9 μm and mammalian cells are 8-20 μm in diameter), making thesize-based separation realizable.

The device is capable of delivering a combined action of (1)sonoporation, (2) electroporation, (3) cell separation/sorting via sizeexclusion, and (4) post-processing cell/DNA transport. Using thesemultiple functions, two complimentary modes of operation can be achievedin microfluidic format:

-   -   Mode 1 (Material Extraction via Cell Lysis)—In this mode the        material is being extracted from the cell by lasing (rupturing)        cell membrane by applying mechanical (acoustic) and/or        electrical force, separately or in combination, of the magnitude        and duration greater than certain threshold values. The        threshold values are determined by calibrating the system.    -   Mode 2 (Material Incorporation via Cell Poration)—In this mode        the material is being injected into the cells by opening the        pores of the cell membrane by applying mechanical (acoustic)        and/or electrical force, separately or in combination, of the        magnitude and duration greater than certain threshold values        required for cell sono- and electro-poration of the cell        membrane, but less than the threshold values leading to cell        lysis, in accordance with the disclosure provided.

This technology is suited for intercellular drug/biomolecule delivery inpharmaceutical, biotech, and clinical applications. Advantages of thetechnology include:

-   -   Combined mechano (sono-) and electro-poration actions.    -   Individual control of transfection on a single-cell level.    -   Simultaneous size-sorting of transfected cells and transport.    -   High throughput & multiplexed operation in microarray format.    -   Small sample volumes & both continuous and discrete operation.    -   Low cost MEMS batch fabrication leading to disposable devices.

The technology has been demonstrated in the laboratory using fluorescentmarkers and mammalian cells. To date, through the proof-of-conceptexperimental studies, we have unambiguously demonstrated theelectrosonic MEMS gun capability for:

-   -   Controllable array operation in drop-on-demand (DOD) mode        desirable for high efficiency cellular transfection.    -   Low power (<100 mW) and temperature (<30° C.) sample ejection        without device clogging by biomolecules/cells and with proven        thermal stability of operation.    -   Flow cytometry results unambiguously indicate that biological        cells remain alive upon processing by the electrosonic MEMS gun.    -   Flow cytometry results unambiguously indicate that biological        cells are able to uptake foreign molecules (e.g., calcein green        fluorescent dye, which do not penetrate the cell membrane under        normal conditions) from the surrounding environment upon        ejection by the electrosonic gun. Thus, use of the electrosonic        gun enables cell treatment which has drug and RNA/DNA/gene        delivery potential.    -   These very promising and significant preliminary results support        the credibility of our approach. Work is on-going to optimize        the operating parameters and device design as well as to test        transfection of different cell-biomolecule combinations.

In one experimental embodiment, an electrosonic DNA gun, according toone embodiment of the invention, was outfitted with an array of 225nozzles with each hole diameter about 35 micrometers. To avoidoverheating of piezoelectric transducer, a pulsing waveform was usedwith a 2-6% duty cycle, 980 kHz driving frequency, and 10 Hz repetitionrate. Three cell samples were analyzed using flow cytometer to determineviability and transfection and uptake efficiency of the device.

A first sample, used for a control experiment, used NIH 3T3 mammaliancells that were used in characterizing device performance and weresuspended in a DMEM medium. Propidium iodide (red fluorescent marker)was then added 10 minutes before cytometry analysis in order to staindead cells. The average cell diameter in suspension was about 15micrometers.

In a second sample used for a viability experiment, an aqueous solutioncontaining cells were ejected by the DNA gun with the flow rate of about100 microliters per minute. Approximately 2 ml of the sample wasprocessed by the device and collected for cytometry analysis. The cellswere then suspended in DMEM medium, propidium iodide (red fluorescentmarker) was added 10 minutes before cytometry analysis in order to staindead cells.

In a third sample used for a transfection/uptake experiment, calcein (agreen fluorescent marker) was added to cell suspension prior to ejectionby the DNA gun. Under normal conditions calcein does not penetrate thecell membrane, i.e., it cannot be incorporated into living cells, so itcan be used to analyze transfection/uptake efficiency of the device. Anaqueous cell suspension containing calcein was ejected by the DNA gun.The sample was collected during 15 minutes of active ejection. Thecollected sample rested for 10 minutes, then cells were centrifuged andthe medium was changed to calcein-free one (through washing). After thatthe propidium iodide (red fluorescent marker) was added 10 minutesbefore cytometry analysis in order to stain dead cells.

These experiments demonstrated the following outcomes:

-   -   Ejection: Biological cells were successfully ejected by the        device without clogging.    -   Viability: Biological cells were shown to remain alive after        being ejected by the device.    -   Transfection: Biological cells were shown to uptake foreign        molecules (i.e., calcein green fluorescent dye) from the        external environment, which do not penetrate the cell membrane        under normal conditions.

The above described embodiments, while including the preferredembodiment and the best mode of the invention known to the inventor atthe time of filing, are given as illustrative examples only. It will bereadily appreciated that many deviations may be made from the specificembodiments disclosed in this specification without departing from thespirit and scope of the invention. Accordingly, the scope of theinvention is to be determined by the claims below rather than beinglimited to the specifically described embodiments above.

1. An apparatus for manipulating cells, comprising: a. a substrate,having a first side and an opposite second side, the substrate definingat least one tapering passage passing therethrough, the tapering passageopening to the first side with a wide end and also opening to the secondside with a narrow end, the narrow end having a size corresponding to asize of a selected cell; b. a first poration electrode and aspaced-apart second poration electrode disposed so as to impart apredetermined electrical field on the passage when an electricalpotential difference is applied between the first poration electrode andthe second poration electrode; c. a fluid driving structure that drivesfluid through the opening, the fluid driving structure including anultrasonic transducer; d. an oscillating circuit that applies anoscillating electric potential to the ultrasonic transducer, therebycausing the ultrasonic transducer to generate a acoustic wave in thetapering passage, wherein the acoustic wave and the electrical fieldimpart energy on at least a portion of the cells so as to cause at leastone of cell poration, cell transfection, cell sorting or cell lysis. 2.The apparatus of claim 1, wherein the ultrasonic transducer is spacedapart from the wide end.
 3. The apparatus of claim 1, wherein theultrasonic transducer is disposed adjacent to the substrate.
 4. Theapparatus of claim 1, wherein the substrate comprises a silicon wafer.5. The apparatus of claim 1, wherein the tapering passage comprises apyramidal passage.
 6. The apparatus of claim 1, wherein the taperingpassage comprises a frusto-conical passage.
 7. The apparatus of claim 1,wherein the first poration electrode and the second poration electrodeeach comprise a conductive substance disposed on an inside surface ofthe tapering passage.
 8. The apparatus of claim 1, wherein the secondporation electrode is disposed adjacent to the second side of thesubstrate.
 9. The apparatus of claim 8, further comprising an insulatorlayer disposed adjacent to the second poration electrode and wherein thefirst poration electrode is disposed adjacent to the insulator layer.10. The apparatus of claim 1, wherein the substrate includes a dopant sothat the substrate comprises the second poration electrode.
 11. Theapparatus of claim 1, wherein the ultrasonic transducer comprises acapacitive transducer.
 12. The apparatus of claim 11, wherein thecapacitive transducer comprises a first transducer electrode andspaced-apart second transducer electrode so that when a potential isapplied between the first transducer electrode and the second transducerelectrode, the second transducer electrode moves relative to the firsttransducer electrode.
 13. The apparatus of claim 1, wherein theultrasonic transducer comprises a piezoelectric transducer.
 14. Theapparatus of claim 13, wherein the piezoelectric transducer comprises alayer of a piezoelectric material disposed between a first transducerelectrode and an opposite second transducer electrode and oriented sothat when an electric field is applied between the first transducerelectrode and the second transducer electrode, a dimensionalcharacteristic of the layer of piezoelectric material changes.
 15. Theapparatus of claim 1, wherein the substrate defines an array of taperingpassages passing therethrough.
 16. The apparatus of claim 15, whereinthe array of tapering passages includes a first plurality of taperingpassages and a spaced-apart second plurality of tapering passages, theapparatus further comprising a wall extending from the substrate so asto separate the first plurality of tapering passages from the secondplurality of tapering passages.
 17. The apparatus of claim 15, whereinthe second poration electrode comprises a plurality of sub-electrodes,wherein each sub-electrode is disposed adjacent to a different set ofthe tapering passages and wherein each sub-electrode may be driven by adifferent electrical potential.